• 한국작물학회지
• /
• 제65권4호
• /
• pp.496-507
• /
• 2020

본 연구는 사과 바이러스 ASGV, ASPV 및 ApMV를 각각 정밀하게 진단하고자 SYBR Green I 및 TaqMan probe, 두 종류의 다른 chemical dye를 사용하여 quantitative real-time PCR 검정법을 개발하고자 하였다. 1. 사과 바이러스 ASGV, ASPV 및 ApMV의 국내분리주를 바탕으로 하여 cloning 및 in vitro transcription을 수행해 10배 희석단위 표준시료를 제작하였다. 각 바이러스에 대한 SYBR Green I용 프라이머와 TaqMan probe용 프라이머 및 프로브를 디자인하였다. 2. 상기 제작된 프라이머와 프로브를 이용해 표준시료를 대상으로 real-time PCR을 수행하여 각 바이러스의 증폭곡선과 검량선을 구할 수 있었다. Real-time PCR 결과, SYBR Green I기반의 검정법은 TaqMan probe기반의 검정법 못지 않은 결과를 보여주었으며, 적은 비용에 대량 검정이 요구되는 곳에 효과적으로 응용될 수 있을 것이다. 3. 현장평가를 본 실험에서 개발된 TaqMan probe기반의 real-time PCR검정법과 기존의 RT-PCR검정법과 비교분석하였다. 그 결과 real-time PCR 검정법은 singleplex 및 multiplex RT-PCR보다 더 민감하고 정확한 결과를 내어 RT-PCR로 검출할 수 없는 농도까지 검정할 수 있음을 입증하였다. 4. 본 실험에서 개발한 real-time PCR검정법은 검역현장과 같은 대량의 검사가 요구되는 곳에서는 SYBR Green I 기반의 검정법을, 바이러스 연구분야에서는 세밀한 정량이 가능한 TaqMan probe 방식의 검정법이 활용될 것으로 기대한다.

• KSBB Journal
• /
• 제29권5호
• /
• pp.361-371
• /
• 2014

Mycoplasma is well recognized as one of the most prevalent and serious microbial contaminants of biologic manufacturing processes. Conventional methods for mycoplasma testing, direct culture method and indirect indicator cell culture method, are lengthy, costly and less sensitive to noncultivable species. In this report, we describe a new TaqMan probe-based real-time PCR method for rapid and quantitative detection of mycoplasma contamination during manufacture of biologics. Universal mycoplasma primers were used for mycoplasma PCR and mycoplasma DNA was quantified by use of a specific TaqMan probe. Specificity, sensitivity, and robustness of the real-time PCR method was validated according to the European Pharmacopoeia. The validation results met required criteria to justify its use as a replacement for the culture method. The established real-time PCR assay was successfully applied to the detection of mycoplasma from human keratinocyte and mesenchymal stem cell as well as Vero cell lines artificially infected with mycoplasma. The overall results indicated that this rapid, specific, sensitive, and robust assay can be reliably used for quantitative detection of mycoplasma contamination during manufacture of biologics.

• 한국동물위생학회지
• /
• 제32권4호
• /
• pp.299-306
• /
• 2009

Assay for the detection and quantification of porcine circovirus type 2 (PCV 2) with the real-time PCR were developed. TaqMan probe real-time using a set of primer/probe was developed for detection of PCV 2. In this study we applied real-time PCR assay to 320 samples, collected from pig farms. In 151 of 320 samples, PCV 2 DNA was detected by conventional PCR assay. All samples positive for PCV 2 DNA in conventional PCR assay were also positive in Real-time PCR assay, but 69 of 169 samples that tested negative for PCV 2 DNA in conventional assay were tested positive in TaqMan probe real-time PCR assay. The test of TaqMan probe real-time PCR resulted in detection and quantification limits of 101 copies per sample. TaqMan probe real-time PCR assay increased the number of samples in which PCV 2 was detected by 21%. TaqMan probe real-time PCR assay is very efficient method in contrast to the conventinal PCR, becoming increasingly important method for gene analysis.

• Journal of Microbiology
• /
• 제41권4호
• /
• pp.320-326
• /
• 2003

In order to identify Vibrio vulnificus in the Yellow Sea near Gunsan, Korea during the early and late summers, the efficiency of the real-time quantitative TaqMan PCR was compared to the efficiency of the conventional PCR and Biolog identification system^TM. Primers and a probe were designed from the hemolysin/cytolysin gene sequence of V. vulnificus strains. The number of positive detections by real-time quantitative TaqMan PCR, conventional PCR, and the Biolog identification system from seawater were 53 (36.8%), 36 (25%), and 10 strains (6.9%), respectively, among 144 samples collected from Yellow Sea near Gunsan, Korea. Thus, the detection method of the real-time quantitative TaqMan PCR assay was more effective in terms of accuracy than that of the conventional PCR and Biolog system. Therefore, our results showed that the real-time TaqMan probe and the primer set developed in this study can be applied successfully as a rapid screening tool for the detection of V. vulnificus.

• Fisheries and Aquatic Sciences
• /
• 제24권11호
• /
• pp.351-359
• /
• 2021

The red sea bream iridovirus (RSIV) belonging to genus Megalocytivirus is responsible for red sea bream iridoviral disease (RSIVD) in marine and freshwater fishes. Although several diagnostic assays for RSIV have been developed, diagnostic sensitivity (DSe) and specificity (DSp) of real-time polymerase chain reaction (PCR) assays are not yet evaluated. In this study, we developed a TaqMan probe-based real-time PCR method and evaluated its DSe and DSp. To detect RSIV, the probe and primers were designed based on consensus sequences of the major capsid protein (MCP) genes from megalocytiviruses including RSIV, infectious spleen and kidney necrosis virus (ISKNV), and turbot reddish body iridovirus (TRBIV). The probe and primers were shown to be specific for RSIV, ISKNV, and TRBIV-types megalocytiviruses. A 95% limit of detection (LOD_95%) was determined to be 5.3 viral genome copies/µL of plasmid DNA containing the MCP gene from RSIV. The DSe and DSp of the developed real-time PCR assay for field samples (n = 112) were compared with those of conventional PCR assays and found to be 100% and 95.2%, respectively. The quantitative results for SYBR Green and TaqMan probe-based real-time PCR were not significantly different. The TaqMan probe-based real-time PCR assay for RSIV may be used as an appropriate diagnostic tool for qualitative and quantitative analysis.

• 대한수의학회지
• /
• 제45권1호
• /
• pp.25-28
• /
• 2005

The melanocortin 1 receptor (MC1R) plays an important role in regulation of melanin pigment synthesis within mammalian melanocytes. Mutations within the gene encoding MC1R have been shown to explain coat color variations within several mammalian species including cattle. To develope a rapid and accurate method for the identification of Hanwoo meat, we performed a single nucleotide polymorphism (SNP) analysis in Melanocortin 1 receptor (MC1R) gene using TaqMan$^{(R)}$ MGB probe-based real-time PCR. Two specific probes (one for Hanwoo and the other for Holstein and Black angus) were designed. At the 5' end of 2 TaqMan$^{(R)}$ MGB probes, 6-carboxyfluorescein (FAM) was labeled for Hanwoo, and VIC for Holstein and Black angus. As a result, Hanwoo samples showed FAM-positive signal only, whereas other samples showed VIC-positive. This result suggests that the TaqMan$^{(R)}$ MGB probe based real-time PCR technique would be very accurate, easy and reproducible method to discriminate between Hanwoo meat and Holstein/Black angus meat.

• Mycobiology
• /
• 제50권5호
• /
• pp.382-388
• /
• 2022

White mold (or Sclerotinia stem rot), caused by Sclerotinia species, is a major air, soil, or seed-transmitted disease affecting numerous crops and wild plants. Microscopic or culture-based methods currently available for their detection and identification are time-consuming, laborious, and often erroneous. Therefore, we developed a multiplex quantitative PCR (qPCR) assay for the discrimination, detection, and quantification of DNA collected from each of the three economically relevant Sclerotinia species, namely, S. sclerotiorum, S. minor, and S. nivalis. TaqMan primer/probe combinations specific for each Sclerotinia species were designed based on the gene sequences encoding aspartyl protease. High specificity and sensitivity of each probe were confirmed for sclerotium and soil samples, as well as pure cultures, using simplex and multiplex qPCRs. This multiplex assay could be helpful in detecting and quantifying specific species of Sclerotinia, and therefore, may be valuable for disease diagnosis, forecasting, and management.

• 한국미생물·생명공학회지
• /
• 제43권3호
• /
• pp.267-274
• /
• 2015

세포주를 이용하여 생산하는 생물의약품과 생산용 세포주는 세포 배양 과정 중에 사용되는 돼지 유래 trypsin으로 부터 외래성 돼지 유래 바이러스가 오염될 가능성이 있다. PTGV는 세포배양 유래 생물의악품 제조공정에서 오염될 수 있는 외래성 바이러스 중의 하나이다. 본 연구에서는 생물의 약품 제조공정에서 PTGV 안전성을 확보하기 위해, 세포주, 원료물질, 제조공정, 완제품에서 PTGV를 정량적으로 검출하고, 제조공정에서 PTGV 제거 검증을 위한 시험법으로 활용이 가능한 TaqMan probe real-time RT-PCR 시험법을 확립하였다. PTGV에 특이적인 primer와 probe를 선별하여 PTGV 정량검출 시험법을 최적화하였다. 세포배양법에 의한 감염역가와 비교한 결과 real-time RT-PCR의 검출한계는 1.10 × 10⁰ TCID₅₀/ml이었다. 확립된 시험법의 신뢰성을 보증하기 위해 시험법 검증을 실시한 결과 특이성과 재현성, 완건성이 우수함을 확인하였다. 확립된 real-time RT-PCR 을 생물의약품 제조공정 검증에 적용할 수 있는지 확인하기 위하여 인위적으로 PTGV를 오염시킨 CHO-K1 세포주에서 PTGV 검출 시험을 실시하였다. PTGV를 감염시킨 CHO-K1 세포에서 세포변병효과를 관찰할 수 없었지만, 세포배양액에서 PTGV를 정량적으로 검출할 수 있었다.

• 미생물학회지
• /
• 제51권3호
• /
• pp.199-208
• /
• 2015

소의 혈액, 세포, 조직, 기관 등이 생물의약품, 조직공학제제, 세포치료제, 의료기기의 원재료로 널리 사용되고 있다. 소유래 성분 원재료에 다양한 바이러스가 오염된 사례가 있기 때문에 소유래 물질을 원재료로 사용한 제제의 바이러스 안전성 검증이 필수적으로 요구된다. Bovine adenovirus type 1(BAdV-1)은 소에게 가장 흔하게 감염되는 바이러스 중의 하나이다. 소유래 물질을 원재료로 하는 생물의약품, 조직공학제제, 세포치료제, 의료기기 등에서 BAdV-1 안전성을 확보하기 위해, 세포주, 원재료, 제조공정, 완제품에서 BAdV-1을 정량적으로 검출하고, 제조공정에서 BAdV-1 제거 검증을 위한 시험법으로 활용이 가능한 TaqMan probe real-time PCR 시험법을 확립하였다. 세포배양법에 의한 감염역가와 비교한 결과, real-time PCR 검출한계는 $7.44{\times}10^1\;TCID_{50}/ml$이었다. 확립 된 시험법의 신뢰성(reliability)을 보증하기 위해 시험법 검증을 실시한 결과, 특이성(specificity)과 재현성(reproducibility), 완건성(robustness)이 우수함을 확인하였다. 확립된 real-time PCR을 생물의약품 제조공정 검증에 적용할 수 있는지 확인한 결과, 인위적으로 BAdV-1을 오염시킨 Chinese Hamster Ovary(CHO) 세포주에서 BAdV-1를 정량적으로 검출할 수 있었다. 확립된 시험법을 항체의약품 생산용 CHO 마스터 세포주와 소유래 type 1 collagen에서 BAdV-1 검출 시험에 산업적으로 적용하였다. 위와 같은 결과에서 확립된 BAdV-1 real-time PCR 시험법은 감염역가 시험법과 같은 생물학적 시험법을 대신할 수 있는 신속하고, 특이성과 민감성이 우수한 시험법임을 확인하였다.

• Journal of Microbiology and Biotechnology
• /
• 제19권11호
• /
• pp.1464-1469
• /
• 2009

To achieve more accurate and rapid detection of macrolide-lincosamide-streptogramin B resistance genes, erm(A), erm(B), and erm(C), we developed a TaqMan probe-based real-time PCR (Q-PCR) method and compared it with conventional PCR (C-PCR), which is the most widely using erm gene identification method. The detection limit of Q-PCR was 5 fg of genomic DNA or 5-8 CFU of bacterial cells of Staphylococcus aureus. The utilization of Q-PCR might shorten the time to erm detection from 3-4 h to about 50 min. These data indicated that Q-PCR assay appears to be not only highly sensitive and specific, but also the most rapid diagnostic method. Therefore, the appropriate application of the Q-PCR assay will permit rapid and accurate identification of erm genes from clinical and other samples.